The global objective of the research proposed here is to understand the fundamental question of how ribozyme catalysis and regulation works, and to apply this understanding to begin to engineer new catalytic properties. Our discovery of ribozymes that regulate gene expression in mammals compels us to understand how RNA structural changes enable switching from a ligated (on) state to a cleaved (off) state, and how the ribozyme's structure gives rise to catalytic activity in its biological context. Using a combination of mechanism-focused X-ray crystallography, single molecule biophysics experiments, and in vitro evolution and selection techniques, we plan to answer three sets of questions that are formulated as the three specific aims of the proposal. The hypothesis that these experiments are designed to test is that the RNA itself forms a dynamic three-dimensional structure that regulates not only its overall catalytic activity, but also regulates a switch between RNA cleavage and RNA ligation. The switch between cleavage and ligation is absolutely critical to understanding both ribozyme-mediated satellite virus replication and a new form of ribozyme-mediated mammalian gene regulation. These specific aims are formulated (1) to answer the question of how the active-site structure of the full-length, natural hammerhead ribozyme enables it to be an enzyme;(2) to understand how a single ribozyme molecule can switch between required nuclease and ligase enzyme activities;and (3) to enable us to engineer new ribozyme functionality, with the ultimate goal of creating a new generation of potentially more potent in vivo ribozyme-based therapeutic agents that target pathogenic RNAs. PUBLIC HEALTH RELEVANCE: The goal of the experiments described in this proposal is to understand how a catalytic RNA called the hammerhead ribozyme can efficiently accelerate an RNA cutting reaction, and how the molecular switch that changes it from an RNA cutting enzyme into an RNA joining enzyme works. By understanding these aspects of ribozyme catalysis, we will better understand satellite RNA virus replication, how ribozyme-mediated gene expression regulation works, and we will be better able to suggest how to design ribozyme therapeutic agents that target RNA viruses that include the cold and flu viruses, hepatitis C and HIV.